ABSTRACT The neurotoxicity of OP pesticides in humans may result from dermal or inhalation exposure on clothing or machinery or orally from the residue on food. In the latter context, the health consequences associated with the use of organophosphate (OP) pesticides, especially in children, could be reduced by monitoring produce and eliminating the consumption of OP-contaminated food. PlantVax currently employs the Nicotiana benthamiana plant expression system to produce high levels of recombinant human acetylcholinesterase (rHuAChE) which is used in devices eg the FLIR CADpen, for the detection of trace amounts of organophosphate (OP) nerve agents for military purposes. In this proposal, PlantVax aims to develop a simplified, hand-held inexpensive PEST-pen to rapidly detect phosphorothionate insecticides on food, clothing and machinery. While the new device will utilize the same esterase-based embedded chemistry as the CADpen with a yellow to red color as a read-out, important features need to be altered to increase the rate of color change efficacy of the new device. Firstly, the inhibition constants (ki) obtained for carboxylases (CES) are higher for insecticides than HuAChE and will initially be incorporated into the pen sponge. Secondly since phosphorothionate insecticides are inactive and need to be metabolically converted by oxidative desulfuration into the active oxon form by cytochrome P450 in the liver for their insecticidal action, the PEST-pen may need to integrate features of both thion oxidation to an oxon as well as CES/AChE inhibition into a single device. The innovation in this proposal will be the identification of novel indicator molecules with high inhibition rates for incorporation into the sponge. These include (i) the wildtype or G137D mutant form of the carboxylesterase enzyme from the Australian blow fly Lucilia cuprina (Lc?E7) which exhibits high kinetic parameters for thion forms or (ii) mutant AChE enzyme with increased rates of inhibition by oxon embedded together with a P450, in the presence of a cytochrome c reductase (NADPH), to expedite conversion to the oxonforms. Based on the design of the successful CADpen, several PEST-pen prototypes with different indicator molecules will be produced and tested within a year.